I start my study of cytokine activation. The whole blood are derived from patient which undergo cardiac surgery operation with the us of Cardiac Pulmonary Bypass. The sample are taken : before anaesthesia, 15 min of CPB, first and second postoperative day. I estimate the IL-4, IL-6, IL-8, MPO, TNF, IFN. I used the BD primary antibody for intracellular staining and DAKO IntraStain for fixation and permabilisation. All the sample are negative. But I?m not sure if the negative results are because there are really no activation of cytokine in this sample or because of wrong procedure. Should I pre-activate the lymphocyte (with BFA, Ionomycine) before the estimation. Many thanks for any suggestion Piotr Wilczek
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